Università degli Studi di Napoli Federico II

176 Pages·2012·4.77 MB·English

Università degli Studi di Napoli Federico II
Dipartimento di Agraria

Ph.D. Thesis in
SCIENZE AGRARIE E AGROALIMENTARI
XXIX cycle (2014-2017)

Metabolomic Fingerprinting of Food Plants by
Nuclear Magnetic Resonance Spectroscopy and Gas
Chromatography/Mass Spectrometry

Supervisor Ph.D. student
Prof.ssa Virginia Lanzotti Bruna de Falco

Coordinator
Prof. Guido D’Urso

Table of contents

Table of contents
1. Introduction ...................................................................................................................... 1
1.1 ‘-Omics’ technologies .......................................................................................................... 1
1.1.1 Genomic and transcriptomic ..................................................................................... 2
1.1.2 Proteomic .................................................................................................................. 3
1.1.3 Metabolomic .............................................................................................................. 3
1.2 Analytical approaches .......................................................................................................... 6
1.3 Data analysis ...................................................................................................................... 10
1.4 Aim of the study ................................................................................................................. 11
References ................................................................................................................................ 13
2. Artichoke: botanical, agronomical, phytochemical and pharmacological overview 17
2.1 Introduction ........................................................................................................................ 17
2.2 Origin and classification ..................................................................................................... 19
2.3 Morphology, ecology and use ............................................................................................ 21
2.4 Nutritional quality .............................................................................................................. 23
2.5 Chemical constituent .......................................................................................................... 23
2.6 Caffeoylquinic acids ........................................................................................................... 24
2.6.1 Biological activity of caffeoylquinic acids .............................................................. 24
2.7 Flavonoids .......................................................................................................................... 29
2.7.1 Biological activity of flavonoids ............................................................................. 31
2.8 Triterpenes and sesquiterpenes ........................................................................................... 34
2.8.1 Biological activity of triterpenes and sesquiterpenes .............................................. 37
2.9 Inulin and its biological activity ......................................................................................... 40
2.10 Comparative analysis of phytochemistry and pharmacology studies .............................. 42
2.10.1 Comparative analysis of studies on artichoke metabolites identification ............. 43
2.10.2 Comparative analysis of studies of metabolites health effects .............................. 46
2.11 Conclusion ........................................................................................................................ 50
References ................................................................................................................................ 52
3. Metabolomic Fingerprinting of Romaneschi Globe Artichokes by NMR
Spectroscopy and Multivariate Data Analysis..................................................................... 61
3.1 Introduction ........................................................................................................................ 61
3.2 Materials and methods ....................................................................................................... 63
3.2.1 Chemicals ................................................................................................................ 63
3.2.2 Plant materials ......................................................................................................... 63
3.2.3 Extraction procedure ............................................................................................... 64
3.2.4 Spectroscopic analysis............................................................................................. 65
3.2.5 Multivariate data analysis ........................................................................................ 66
3.3 Results and discussions ...................................................................................................... 66
3.3.1 Metabolite profile .................................................................................................... 66
3.3.2 Organic acids ........................................................................................................... 67
3.3.3 Free amino acids ...................................................................................................... 68
3.3.4 Carbohydrates .......................................................................................................... 73
3.3.5 Polyphenols ............................................................................................................. 73
3.3.6 Flavonoids ............................................................................................................... 75
3.3.7 Terpenoid glycosides ............................................................................................... 76
Table of contents

3.3.8 PCA results .............................................................................................................. 76
3.4 Conclusion .......................................................................................................................... 80
References ................................................................................................................................ 84
4. Chia seeds products: an overview ................................................................................. 87
4.1 Introduction ........................................................................................................................ 87
4.2 Chemical constituent .......................................................................................................... 88
4.3 Caffeic acid derivatives ...................................................................................................... 89
4.4 Flavonoids .......................................................................................................................... 90
4.5 Oil composition .................................................................................................................. 94
4.6 Fibers ................................................................................................................................ 100
4.7 Total polyphenolic content and their antioxidant activity ................................................ 102
4.8 Industrial uses ................................................................................................................... 103
4.9 Conclusion ........................................................................................................................ 105
References .............................................................................................................................. 106
5. Metabolomic Analysis of Salvia hispanica Seeds Using NMR Spectroscopy and
Multivariate Data Analysis .................................................................................................. 113
5.1 Introduction ...................................................................................................................... 113
5.2 Materials and methods ..................................................................................................... 115
5.2.1 Chemicals .............................................................................................................. 115
5.2.2 Seeds obtained with different nitrogen supply ...................................................... 116
5.2.3 Commercial and long-day flowering chia ............................................................. 116
5.2.4 Extraction procedure ............................................................................................. 117
5.2.5 Spectroscopic analysis........................................................................................... 117
5.2.6 Multivariate data analysis ...................................................................................... 118
5.2.7 Chemometrics ........................................................................................................ 119
5.3 Results and discussions .................................................................................................... 120
5.3.1 Metabolite profile .................................................................................................. 120
5.3.2 Organic acids ......................................................................................................... 120
5.3.3 Free amino acids .................................................................................................... 122
5.3.4 Carbohydrates ........................................................................................................ 122
5.3.5 Flavonoids ............................................................................................................. 124
5.3.6 Polyphenols ........................................................................................................... 124
5.3.7 Other compounds .................................................................................................. 126
5.3.8 Fatty acids ............................................................................................................. 126
5.3.9 Exploratory multivariate analysis .......................................................................... 127
5.3.10 Chemometrics for single metabolites .................................................................. 130
5.3.11 Effects of mineral fertilization ............................................................................ 132
5.4 Conclusion ........................................................................................................................ 136
References .............................................................................................................................. 138
6. Metabolomic Analysis by UAE-GC MS and antioxidant activity of Salvia hispanica
seeds grown under different irrigation regimes ................................................................ 143
6.1 Introduction ...................................................................................................................... 143
6.2 Materials and methods ..................................................................................................... 146
6.2.1 Chemicals .............................................................................................................. 146
6.2.2 Plant materials ....................................................................................................... 146
6.2.3 Growth conditions ................................................................................................. 146
6.2.4 Extraction procedure of the organic phase ............................................................ 147
Table of contents

6.2.5 Ultrasound assisted extraction (UAE) ................................................................... 147
6.2.6 Total polyphenol content ....................................................................................... 148
6.2.7 Antioxidant activity ............................................................................................... 148
6.2.8 Derivatization and GC/MS analysis ...................................................................... 149
6.2.9 Statistical analysis ................................................................................................. 150
6.3 Results and discussions .................................................................................................... 150
6.3.1 Seeds production and chemical properties ............................................................ 150
6.3.2 Antioxidant activity ............................................................................................... 151
6.3.3 Metabolite profile .................................................................................................. 152
6.3.4 Apolar phase .......................................................................................................... 153
6.3.5 Polar phase ............................................................................................................ 156
6.4 Conclusion ........................................................................................................................ 160
References .............................................................................................................................. 161
7. General conclusion ....................................................................................................... 167

When we administer medicine, we administer the whole world:
that is, all the virtue of heaven and earth, air and water.
Therefore, your medicine must contain the whole firmament of
both upper and lower spheres. Think with what energy the
Nature takes heaven and earth with all their powers when she
strives against death.

Theophrastus Bombastus von Hohenheim, 1530: Das Buch
Paragranum

Chapter 1 Introduction

1. Introduction

1.1 ‘-Omics’ technologies

Over the past few years, the ‘-omics’ fields have seen an explosive growth opening new
perspectives for biological research purpose. The development of analytical instrumentations,
data processing and chemometric tools simplify the study of complex biological systems on a
large-scale. Metabolomic, together with other ‘-omics’ disciplines such as genomic,
transcriptomic, and proteomic, is becoming an integral part of a system biological approach for
investigating organisms. Fig. 1.1 reports the classification of the ‘-omics’ technologies and the
correlation among them. Although transcriptome represents the process for protein synthesis,
an increase in mRNA levels does not always correspond with an increase in proteins due to
numerous post-transcriptional regulation mechanisms (Kendrick 2014; Vogel et al. 2012).
Therefore, changes in transcriptome or proteome do not always reflect alterations in
biochemical phenotypes. For this reason, the association of metabolomic with the other
analytical areas of genomic, transcriptomic and proteomic constitute a very powerful method
to study biological systems.
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Chapter 1 Introduction

Fig. 1.1 General classification of ‘-omics’ technologies

1.1.1 Genomic and transcriptomic
Genomic is considered a discipline of genetics able to define the overall genome of organisms,
that includes genes, noncoding DNA and also the genomes from mitochondria and chloroplasts.
On the other side, transcriptomic aims to determine the expression level of genes (mRNA and
noncoding mRNA). Genome and transcriptome consist of linear polymers of five nucleotides
with highly similar chemical properties. Several technologies have been developed to determine
and quantify simultaneously a high number of genes and significant changes of their mRNA.
In the late 90’s and 2000’s, DNA array technology progressed rapidly as a new method in which
the hybridization plays an important role to measure the expression levels of genes (Bumgarner
2013). More recently, sequence-based approaches directly determine the cDNA sequences and
the changing of the cellular transcriptome. Although RNA-seq is a tool still under development,
it offers several advantages, such as the use of low amount of RNA, the ability to distinguish
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